LDL-mediated interaction of Lp[a] with HepG2 cells: a novel fluorescence microscopy approach.
We studied the topography of Lp[a]-LDL-cell interactions by means of fluorescence microscopy, using fluorescence-labeled lipoproteins. In contrast to known methods which are based on noncovalent labeling of lipoproteins by positively charged amphiphiles, the protein moiety of LDL and Lp[a] was covalently labeled with either BODIP-succinimide-ester (green) or rhodamine X iodoacetamide (red). The interaction of the fluorescent lipoproteins with cultured HepG2 cells was studied using a confocal laser scanning fluorescence microscope. LDL and Lp[a], each labeled with a different dye, could be examined separately within a mixture of both lipoproteins during their interaction with HepG2 cells. At 4 degrees C, the majority of both fluorescent particles co-localized and only a few separate LDL- or Lp[a]-binding domains could be observed. Quantification of the amount of fluorescent lipoprotein associated with the cell surface at 4 degrees C showed that binding of Lp[a] was increased in the presence of LDL under these conditions, probably via formation of an Lp[a]-LDL complex. At 37 degrees C, LDL and Lp[a] were taken up by the cells within 10 min. Again the majority of LDL and Lp[a] particles co-localized intracellularly. Only minor amounts of LDL and Lp[a] could be observed separately. As the entire fluorescence of labeled Lp[a] co-localized with excess of LDL in cells, and taking into account the high tendency of LDL-Lp[a] association in solution and on cell surfaces, it is concluded that a significant portion of the internalized Lp[a] is taken up into the cells by the LDL receptor via LDL by a hitchhiking-like process.
Hofer
G
G
Department of Biochemistry and Food Chemistry, Technische Universität Graz, Austria.
Steyrer
E
E
Kostner
G M
GM
Hermetter
A
A
D000818:Animals; D001896:Boron Compounds / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D002417:Cattle; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D017270:Lipoprotein(a) / Q000032:analysis / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000032:analysis / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D011485:Protein Binding; D011973:Receptors, LDL / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D017270:Lipoprotein(a); D008077:Lipoproteins, LDL; D008565:Membrane Proteins; D011973:Receptors, LDL; D012235:Rhodamines